Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity.

Immobilization on solid supports provides an effective way to improve enzyme stability and simplify downstream processing for biotechnological applications, which has been widely used in research and in applications. However, surface immobilization may disrupt enzyme structure due to interactions between the enzyme and the supporting substrate, leading to a loss of the enzyme catalytic efficiency and stability. Here, we use a model enzyme, nitroreductase (NfsB), to demonstrate that engineered variants with two strategically positioned surface-tethering sites exhibit improved enzyme stability when covalently immobilized onto a surface. Tethering sites were designed based on molecular dynamics (MD) simulations, and enzyme variants containing cysteinyl residues at these positions were expressed, purified, and immobilized on maleimide-terminated self-assembled monolayer (SAM) surfaces. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the NfsB enzyme orientations, which were found to be consistent with those predicted from the MD simulations. Thermal stability analyses demonstrated that NfsB variants immobilized through two tethering sites exhibited generally improved thermal stability compared with enzymes tethered at only one position. For example, NfsB enzyme chemically immobilized via positions 423 and 111 exhibits at least 60% stability increase compared to chemically immobilized NfsB mutant via a single site. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability.

Monitoring Antimicrobial Mechanisms of Surface-Immobilized Peptides in Situ.

Antimicrobial peptides (AMPs) in free solution can kill bacteria by disrupting bacterial cell membranes. Their modes of action have been extensively studied, and various models ranging from pore formation to carpet-like mechanisms were proposed. Surface-immobilized AMPs have been used as coatings to kill bacteria and as sensors to capture bacteria, but the interaction mechanisms of surface-immobilized AMPs and bacteria are not fully understood. In this research, an analytical platform, sum frequency generation (SFG) microscope, which is composed of an SFG vibrational spectrometer and a fluorescence microscope, was used to probe molecular interactions between surface-immobilized AMPs and bacteria in situ in real time at the solid/liquid interface. SFG probed the molecular structure of surface-immobilized AMPs while interacting with bacteria, and fluorescence images of dead bacteria were monitored as a function of time during the peptide-bacteria interaction. It was believed that upon bacteria contact, the surface-immobilized peptides changed their orientation and killed bacteria. This research demonstrated that the SFG microscope platform can examine the structure and function (bacterial killing) at the same time in the same sample environment, providing in-depth understanding on the structure-activity relationships of surface-immobilized AMPs.

Chemically Immobilized Antimicrobial Peptide on Polymer and Self-Assembled Monolayer Substrates.

Surfaces with chemically immobilized antimicrobial peptides have been shown to have great potential in various applications such as biosensors and antimicrobial coatings. This research investigated the chemical immobilization of a cecropin-melittin hybrid antimicrobial peptide on two different surfaces, a polymer surface prepared by chemical vapor deposition (CVD) polymerization and a self-assembled monolayer surface. We probed the structure of immobilized peptides using spectroscopic methods and correlated such structural information to the measured antimicrobial activity. We found that the hybrid peptide adopts an α-helical structure after immobilization onto both surfaces. As we have shown previously for another α-helical peptide, MSI-78, immobilized on a SAM, we found that the α-helical hybrid peptide lies down when it contacts bacteria. This study shows that the antimicrobial activity of the surface-immobilized peptides on the two substrates can be well explained by the spectroscopically measured peptide structural data. In addition, it was found that the polymer-based antimicrobial peptide coating is more stable. This is likely due to the fact that the SAM prepared using silane may be degraded after several days whereas the polymer prepared by CVD polymerization is more stable than the SAM, leading to a more stable antimicrobial coating.

Simultaneous Observation of the Orientation and Activity of Surface-Immobilized Enzymes.

Surface immobilized enzymes have been widely used in many applications such as biosensors, biochips, biofuel production, and biofuel cell construction. Many factors dictate how enzymes' structure, activity, and stability may change when immobilized, including surface functionalization, immobilization chemistry, nature of the solid support, and enzyme surface density. To better understand how immobilization affects enzyme structure and activity, we have developed a method to measure both surface-sensitive protein vibrational spectra and enzymatic activity simultaneously. To accomplish this, an optical/fluorescence microscope was incorporated into a sum frequency generation (SFG) spectrometer. Using ß-glucosidase (ß-Glu) as a model system, enzymes were covalently tethered to a self-assembled monolayer surface using cysteine-maleimide chemistry. Their orientations were determined by SFG spectroscopy, with a single native cysteine residue oriented toward the functionalized surface, and activity measured simultaneously using a fluorogenic substrate resorufin ß-d-glucopyranoside, with a loss of activity of 53% as compared to comparable solution measurements. Measuring ß-Glu activity and orientation simultaneously provides more accurate information for designing and further improving enzymatic activity of surface-bound enzymes.

Effect of immobilization site on the orientation and activity of surface-tethered enzymes.

Tethering peptides and proteins to abiotic surfaces has the potential to create biomolecule-functionalized surfaces with useful properties. Commonly used methods of immobilization lack control over the orientation in which biological molecules are covalently or physically bound to the surface, leading to sub-optimal materials. Here we use an engineered beta-galactosidase that can be chemically immobilized on a surface with a well-defined orientation through unique surface-accessible cysteine residues. A combined study using sum frequency generation (SFG) vibrational spectroscopy and coarse grained molecular dynamics (MD) simulations was performed to determine the effects of enzyme immobilization site and abiotic surface chemistry on enzyme surface orientation, surface coverage, and catalytic activity. Two beta-galactosidase variants that were immobilized through cysteine introduced at positions 227 and 308 were studied. In both cases, when the abiotic surface was made more hydrophilic, the enzyme surface coverage decreased, but the activity increased. MD simulations indicated that this is due to the weakened interactions between the immobilized enzyme and the more hydrophilic surface. These studies provide improved understanding of how enzyme-surface interactions can be optimized to maximize the catalytic activity of surface tethered enzymes.

Effect of Interfacial Molecular Orientation on Power Conversion Efficiency of Perovskite Solar Cells.

A wide variety of charge carrier dynamics, such as transport, separation, and extraction, occur at the interfaces of planar heterojunction solar cells. Such factors can affect the overall device performance. Therefore, understanding the buried interfacial molecular structure in various devices and the correlation between interfacial structure and function has become increasingly important. Current characterization techniques for thin films such as X-ray diffraction, cross section scanning electronmicroscopy, and UV-visible absorption spectroscopy are unable to provide the needed molecular structural information at buried interfaces. In this study, by controlling the structure of the hole transport layer (HTL) in a perovskite solar cell and applying a surface/interface-sensitive nonlinear vibrational spectroscopic technique (sum frequency generation vibrational spectroscopy (SFG)), we successfully probed the molecular structure at the buried interface and correlated its structural characteristics to solar cell performance. Here, an edge-on (normal to the interface) polythiophene (PT) interfacial molecular orientation at the buried perovskite (photoactive layer)/PT (HTL) interface showed more than two times the power conversion efficiency (PCE) of a lying down (tangential) PT interfacial orientation. The difference in interfacial molecular structure was achieved by altering the alkyl side chain length of the PT derivatives, where PT with a shorter alkyl side chain showed an edge-on interfacial orientation with a higher PCE than that of PT with a longer alkyl side chain. With similar band gap alignment and bulk structure within the PT layer, it is believed that the interfacial molecular structural variation (i.e., the orientation difference) of the various PT derivatives is the underlying cause of the difference in perovskite solar cell PCE.

Molecular Interactions between Graphene and Biological Molecules.

Applications of graphene have extended into areas of nanobio-technology such as nanobio-medicine, nanobio-sensing, as well as nanoelectronics with biomolecules. These applications involve interactions between proteins, peptides, DNA, RNA etc. and graphene, therefore understanding such molecular interactions is essential. For example, many applications based on using graphene and peptides require peptides to interact with (e.g., noncovalently bind to) graphene at one end, while simultaneously exposing the other end to the surrounding medium (e.g., to detect analytes in solution). To control and characterize peptide behavior on a graphene surface in solution is difficult. Here we successfully probed the molecular interactions between two peptides (cecropin P1 and MSI-78(C1)) and graphene in situ and in real-time using sum frequency generation (SFG) vibrational spectroscopy and molecular dynamics (MD) simulation. We demonstrated that the distribution of various planar (including aromatic (Phe, Trp, Tyr, and His)/amide (Asn and Gln)/Guanidine (Arg)) side-chains and charged hydrophilic (such as Lys) side-chains in a peptide sequence determines the orientation of the peptide adsorbed on a graphene surface. It was found that peptide interactions with graphene depend on the competition between both planar and hydrophilic residues in the peptide. Our results indicated that part of cecropin P1 stands up on graphene due to an unbalanced distribution of planar and hydrophilic residues, whereas MSI-78(C1) lies down on graphene due to an even distribution of Phe residues and hydrophilic residues. With such knowledge, we could rationally design peptides with desired residues to manipulate peptide-graphene interactions, which allows peptides to adopt optimized structure and exhibit excellent activity for nanobio-technological applications. This research again demonstrates the power to combine SFG vibrational spectroscopy and MD simulation in studying interfacial biological molecules.

Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy.

Understanding molecular structures of interfacial peptides and proteins impacts many research fields by guiding the advancement of biocompatible materials, new and improved marine antifouling coatings, ultrasensitive and highly specific biosensors and biochips, therapies for diseases related to protein amyloid formation, and knowledge on mechanisms for various membrane proteins and their interactions with ligands. Developing methods for measuring such unique systems, as well as elucidating the structure and function relationship of such biomolecules, has been the goal of our lab at the University of Michigan. We have made substantial progress to develop sum frequency generation (SFG) vibrational spectroscopy into a powerful technique to study interfacial peptides and proteins, which lays a foundation to obtain unique and valuable insights when using SFG to probe various biologically relevant systems at the solid/liquid interface in situ in real time. One highlighting feature of this Account is the demonstration of the power of combining SFG with other techniques and methods such as ATR-FTIR, surface engineering, MD simulation, liquid crystal sensing, and isotope labeling in order to study peptides and proteins at interfaces. It is necessary to emphasize that SFG plays a major role in these studies, while other techniques and methods are supplemental. The central role of SFG is to provide critical information on interfacial peptide and protein structure (e.g., conformation and orientation) in order to elucidate how surface engineering (e.g., to vary the structure) can ultimately affect surface function (e.g., to optimize the activity). This Account focuses on the most significant recent progress in research on interfacial peptides and proteins carried out by our group including (1) the development of SFG analysis methods to determine orientations of regular as well as disrupted secondary structures, and the successful demonstration and application of an isotope labeling method with SFG to probe the detailed local structure and microenvironment of peptides at buried interfaces, (2) systematic research on cell membrane associated peptides and proteins including antimicrobial peptides, cell penetrating peptides, G proteins, and other membrane proteins, discussing the factors that influence interfacial peptide and protein structures such as lipid charge, membrane fluidity, and biomolecule solution concentration, and (3) in-depth discussion on solid surface immobilized antimicrobial peptides and enzymes. The effects of immobilization method, substrate surface, immobilization site on the peptide or protein, and surrounding environment are presented. Several examples leading to high impact new research are also briefly introduced: The orientation change of alamethicin detected while varying the model cell membrane potential demonstrates the feasibility to apply SFG to study ion channel protein gating mechanisms. The elucidation of peptide secondary structures at liquid crystal interfaces shows promising results that liquid crystal can detect and recognize different peptides and proteins. The method of retaining the native structure of surface immobilized peptides or proteins in air demonstrates the feasibility to protect and preserve such structures via the use of hydromimetic functionalities when there is no bulk water. We hope that readers in many different disciplines will benefit from the research progress reported in this Account on SFG studies of interfacial structure-function relationships of peptides and proteins and apply this powerful technique to study interfacial biomolecules in the future.

Capsaicin-Inspired Thiol-Ene Terpolymer Networks Designed for Antibiofouling Coatings.

Novel photocurable ternary polymer networks were prepared by incorporating N-(4-hydroxy-3-methoxybenzyl)-acrylamide (HMBA) into a cross-linked thiol-ene network based on poly(ethylene glycol)diacrylate (PEGDA) and (mercaptopropyl)methylsiloxane homopolymers (MSHP). The ternary network materials displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and reduced the attachment of marine organism Phaeodactylum tricornutum. Extensive soaking of the polymer networks in aqueous solution indicated that no active antibacterial component leached out of the materials, and thus the ternary thiol-ene coating killed the bacteria by surface contact. The surface structures of the polymer networks with varied content ratios were studied by sum frequency generation (SFG) vibrational spectroscopy. The results demonstrated that the PDMS Si-CH3 groups and mimic-capsaicine groups are predominantly present at the polymer-air interface of the coatings. Surface reorganization was apparent after polymers were placed in contact with D2O: the hydrophobic PDMS Si-CH3 groups left the surface and returned to the bulk of the polymer networks, and the hydrophilic PEG chains cover the polymer surfaces in D2O. The capasaicine methoxy groups are able to segregate to the surface in an aqueous environment, depending upon the ratio of HMBA/PEGDA. SFG measurements in situ showed that the antibacterial HMBA chains, rather than the nonfouling PEG, played a dominant role in mediating the antibiofouling performance in this particular polymer system.

Live-cell quantification and comparison of mammalian oocyte cytosolic lipid content between species, during development, and in relation to body composition using nonlinear vibrational microscopy.

Cytosolic lipids participate in the growth, development, and overall health of mammalian oocytes including many roles in cellular homeostasis. Significant emphasis has been placed on the study of lipids as a dynamic organelle, which in turn requires the development of tools and techniques to quantitate and compare how lipid content relates to cellular structure, function, and normalcy. Objectives of this study were to determine if nonlinear vibrational microscopy (e.g., coherent anti-Stokes Raman scattering or CARS microscopy) could be used for live-cell imaging to quantify and compare lipid content in mammalian oocytes during development and in relation to body composition; and compare its efficacy to methods involving cellular fixation and staining protocols. Results of this study demonstrate that CARS is able to identify lipids in live mammalian oocytes, and there exists quantifiable and consistent differences in percent lipid composition across ooctyes of different species, developmental stages, and in relation to body composition. Such a method of live-cell lipid quantification has (i) experimental power in basic cell biology, (ii) practical utility for identifying developmental predictive biomarkers while advancing biology-based oocyte/embryo selection, and (iii) ability to yield rationally supporting technology for decision-making in rodents, domestic species, and human assisted reproduction and/or fertility preservation.

Influence of the side chain and substrate on polythiophene thin film surface, bulk, and buried interfacial structures.

The molecular structures of organic semiconducting thin films mediate the performance of various devices composed of such materials. To fully understand how the structures of organic semiconductors alter on substrates due to different polymer side chains and different interfacial interactions, thin films of two kinds of polythiophene derivatives with different side-chains, poly(3-hexylthiophene) (P3HT) and poly(3-potassium-6-hexanoate thiophene) (P3KHT), were deposited and compared on various surfaces. A combination of analytical tools was applied in this research: contact angle goniometry and X-ray photoelectron spectroscopy (XPS) were used to characterize substrate dielectric surfaces with varied hydrophobicity for polymer film deposition; X-ray diffraction and UV-vis spectroscopy were used to examine the polythiophene film bulk structure; sum frequency generation (SFG) vibrational spectroscopy was utilized to probe the molecular structures of polymer film surfaces in air and buried solid/solid interfaces. Both side-chain hydrophobicity and substrate hydrophobicity were found to mediate the crystallinity of the polythiophene film, as well as the orientation of the thiophene ring within the polymer backbone at the buried polymer/substrate interface and the polymer thin film surface in air. For the same type of polythiophene film deposited on different substrates, a more hydrophobic substrate surface induced thiophene ring alignment with the surface normal at both the buried interface and on the surface in air. For different films (P3HT vs. P3KHT) deposited on the same dielectric substrate, a more hydrophobic polythiophene side chain caused the thiophene ring to align more towards the surface at the buried polymer/substrate interface and on the surface in air. We believe that the polythiophene surface, bulk, and buried interfacial molecular structures all influence the hole mobility within the polythiophene film. Successful characterization of an organic conducting thin film surface, buried interfacial, and bulk structures is a first crucial step in understanding the structure-function relationship of such films in order to optimize device performance. An in-depth understanding on how the side-chain influences the interfacial and surface polymer orientation will guide the future molecular structure design of organic semiconductors.

Multireflection sum frequency generation vibrational spectroscopy.

We developed a multireflection data collection method in order to improve the signal-to-noise ratio (SNR) and sensitivity of sum frequency generation (SFG) spectroscopy, which we refer to as multireflection SFG, or MRSFG for short. To achieve MRSFG, a collinear laser beam propagation geometry was adopted and trapezoidal Dove prisms were used as sample substrates. An in-depth discussion on the signal and SNR in MRSFG was performed. We showed experimentally, with "m" total internal reflections in a Dove prism, MRSFG signal is ∼m times that of conventional SFG; SNR of the SFG signal-to-background is improved by a factor of >m(1/2) and

Effect of Solvent on Surface Ordering of Poly(3-hexylthiophene) Thin Films.

Enhancement of charge transport in organic polymer semiconductors is a crucial step in developing optimized devices. A variety of sample preparation conditions, such as film fabrication method, solvent species, and annealing, were found to influence the hole mobility of organic polymers. Despite the fact that many factors can influence their performance, it is believed that polymer surface ordering plays a key role in determining organic polymer function. Here, sum frequency generation (SFG) vibrational spectroscopy was used to nondestructively map the surface/interfacial ordering of poly(3-hexylthiophene) (P3HT) films prepared using different solvents; we believe that solvent interactions determine the degree of surface/interfacial ordering. Both X-ray diffraction (XRD) spectroscopy and scanning electron microscopy (SEM) were used to supplement SFG to systematically study bulk crystallinity and surface morphology. We conclude that SFG is a powerful tool to elucidate the surface/interfacial structural information on polymer semiconducting films. We demonstrate that the solvent composition used to prepare P3HT thin films influences the resulting film surface morphology, surface/interfacial ordering, and bulk crystallinity.

Sum frequency generation vibrational spectroscopic studies on buried heterogeneous biointerfaces.

A sum frequency generation (SFG) vibrational micro-spectroscopy system was developed to examine buried heterogeneous biointerfaces. A compact optical microscope was constructed with total-internal reflection (TIR) SFG geometry to monitor the tightly focused SFG laser spots on interfaces, providing the capability of selectively probing different regions on heterogeneous biointerfaces. The TIR configuration ensures and enhances the SFG signal generated only from the sample/substrate interfacial area. As an example for possible applications in biointerfaces studies, the system was used to probe and compare buried interfacial structures of different biological samples attached to underwater surfaces. We studied the interface of a single mouse oocyte on a silica prism to demonstrate the feasibility of tracing and studying a single live cell and substrate interface using SFG. We also examined the interface between a marine mussel adhesive plaque and a CaF2 substrate, showing the removal of interface-bonded water molecules. This work also paves the way for future integration of other microscopic techniques such as TIR-fluorescence microscopy or nonlinear optical imaging with SFG spectroscopy for multimodal surface or interface studies.

Peering beneath the surface: novel imaging techniques to noninvasively select gametes and embryos for ART.

Embryo imaging has long been a critical tool for in vitro fertilization laboratories, aiding in morphological assessment of embryos, which remains the primary tool for embryo selection. With the recent emergence of clinically applicable real-time imaging systems to assess embryo morphokinetics, a renewed interest has emerged regarding noninvasive methods to assess gamete and embryo development as a means of inferring quality. Several studies exist that utilize novel imaging techniques to visualize or quantify intracellular components of gametes and embryos with the intent of correlating localization of organelles or molecular constitution with quality or outcome. However, the safety of these approaches varies due to the potential detrimental impact of light exposure or other variables. Along with complexity of equipment and cost, these drawbacks currently limit clinical application of these novel microscopes and imaging techniques. However, as evidenced by clinical incorporation of some real-time imaging devices as well as use of polarized microscopy, some of these imaging approaches may prove to be useful. This review summarizes the existing literature on novel imaging approaches utilized to examine gametes and embryos. Refinement of some of these imaging systems may permit clinical application and serve as a means to offer new, noninvasive selection tools to improve outcomes for various assisted reproductive technology procedures.

Cell volume changes during apoptosis monitored in real time using digital holographic microscopy.

Cellular volume changes play important roles in many processes associated with the normal cell activity, as well as various diseases. Consequently, there is a considerable need to accurately measure volumes of both individual cells and cell populations as a function of time. In this study, we have monitored cell volume changes in real time during apoptosis using digital holographic microscopy. Cell volume changes were deduced from the measured phase change of light transmitted through cells. Our digital holographic experiments showed that after exposure to 1 µM staurosporine for 4 h, the volumes of KB cells were reduced by ~50-60%, which is consistent with previous results obtained using electronic cell sizing and atomic force microscopy. In comparison with other techniques, digital holographic microscopy is advantageous because it employs noninvasive detection, has high time resolution, real time measurement capability, and the ability to simultaneously investigate time-dependent volume changes of both individual cells and cell populations.

Observing a model ion channel gating action in model cell membranes in real time in situ: membrane potential change induced alamethicin orientation change.

Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split α/3(10)-helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer and was measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH = 6.7 the α-helix at the N-terminus and the 3(10)-helix at the C-terminus tilt at about 72° (θ(1)) and 50° (θ(2)) versus the surface normal, respectively. When pH increases to 11.9, θ(1) and θ(2) decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ(1) for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and the bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes.

Molecular interactions of proteins and peptides at interfaces studied by sum frequency generation vibrational spectroscopy.

Interfacial peptides and proteins are critical in many biological processes and thus are of interest to various research fields. To study these processes, surface sensitive techniques are required to completely describe different interfacial interactions intrinsic to many complicated processes. Sum frequency generation (SFG) spectroscopy has been developed into a powerful tool to investigate these interactions and mechanisms of a variety of interfacial peptides and proteins. It has been shown that SFG has intrinsic surface sensitivity and the ability to acquire conformation, orientation, and ordering information about these systems. This paper reviews recent studies on peptide/protein-substrate interactions, peptide/protein-membrane interactions, and protein complexes at interfaces and demonstrates the ability of SFG on unveiling the molecular pictures of complicated interfacial biological processes.

Examining surface and bulk structures using combined nonlinear vibrational spectroscopies.

We combined sum-frequency generation (SFG) vibrational spectroscopy with coherent anti-Stokes Raman scattering (CARS) spectroscopy in one system to examine both surface and bulk structures of materials with the same geometry and without the need to move the sample. Poly(methyl methacrylate) (PMMA) and polystyrene (PS) thin films were tested before and after plasma treatment. The sensitivities of SFG and CARS were tested by varying polymer film thickness and using a lipid monolayer.

Calibration approach for fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation.

Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.